以單核苷酸多型性之分析應用於裂解DNA人別鑑定之研究

Abstract

在法醫學上進行人別鑑定之技術，目前是以PCR為基礎的短相連重複序列(STR)為主，但是STR對於分析腐敗DNA(degraded DNA)檢體的能力有限。而目前許多研究指出單核苷酸多型性(SNP)分析可以有效使用於腐敗DNA型別檢測，因此本研究目的主要是要設計一套適合應用於腐敗檢體的SNP分析套組 本研究收集16個台灣漢人血液檢體以及17個台灣漢人胎盤組織檢體(包括2個家族的3個3人親子組及1個2人親子組)，萃取DNA後使用DNase I酵素法作用30及60分鐘，將DNA進行裂解以模擬法醫檢體腐敗DNA的片段長度(<100bp)，使用即時定量聚合酶連鎖反應(qPCR)進行DNA定量，並以定量結果進行STR基因座分析，分析結果若STR圖形訊號由短片段至長片段逐漸遞減，甚至是長片段基因座消失，則確定其為裂解DNA檢體之STR圖形，且無法完整得知STR基因座之訊息。另以電泳生物分析(Bioanalyzer)來觀察裂解DNA片段分布，發現本研究所製作之裂解DNA片段分布在15bp以下，再把這些確定為裂解DNA之檢體，使用產物長度為60~100bp的145個體染色體SNP點位以基質輔助雷射脫附游離飛行時間質譜儀(MALDI–TOF)的方法進行SNP分析。 研究結果顯示此145個SNP點位在30分鐘及60分鐘之裂解DNA檢體平均成功率各為46%以及60%，其中51個點位在30分鐘及60分鐘裂解檢體之成功分析率達50%以上，可做裂解檢體之分析。145個SNP點位於研究族群中的哈溫平衡值皆無顯著差異，而鑑別力(power of discrimination)可達0.9999999999，累積三人排除率(CPEtrio)達0.9999999999，累積兩人排除率(CPEduo)則是達到0.999999。以研究族群中的家族實例計算，無法以STR分析之裂解檢體可以使用SNP分析進行親子鑑定。我們的結論是，本研究所使用的SNP系統能分析裂解DNA檢體的SNP型別及協助人別鑑定。

Forensic genetics has been currently using PCR based Short Tandem Repeat (STR) polymorphisms. However, STR can be non-informative when DNA samples are degraded. Recent advances in single nucleotide polymorphisms (SNPs) research have raised the possibility that the SNP markers could replace the STR in analyzing degraded DNA samples. The purpose of this study was to develope an effective SNPs panel to genotype degraded DNA samples. We collected 16 blood samples and 17 pacenta samples of Han Taiwanese (including 2 families with 3 trio and 1 duo sets). DNA was extracted and degraded to <100bp by DNase I treatment. Then qPCR quantification and STR genotyping were performed. The STR typing of degraded DNA showed gradually degradation signal pattern from shorter size STR loci to longer size, eventhough disappeared. The DNA fragments distribution was analyzed using Bioanalyzer and the length of DNA fragments revealed below 15 bp. Finally, a panel including 145 autosomal SNP loci was used to analyze degraded DNA by matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS). The target amplicons for each 145 SNPs arranged from 60 bp to 100 bp. Among the 145 SNP loci, 46% and 60% of genotypes could be successfully identified in 30 min and 60 min degeaded DNA samples. There were 51 SNP loci with detection rate above 50% for both 30min and 60min degraded samples. The total power of discrimination (Pd) was 0.9999999999, the cumulative probability of exclusion (CPEtrio) of trios-testing was 0.9999999999, and the cumulative probability of exclusion of duo-testing (CPEduo) was 0.999999. Compared to STR loci, these SNP loci offered a more informative analysis for degraded DNA in real parentage testing. In conclusion, this 145 SNP loci panel can be helpful in the analysis of degraded DNA and individual identification.